It is thought that defects in the process of B lymphocyte tolerance are central to the pathogenesis of systemic lupus erythernatosus (SLE). Moreover, the importance of B cell receptor (BCR) signal strength for the regulation of tolerance is evident from murine models of autoimmunity which suggest that specific defects in lymphocyte signaling can lead to SLE-like phenotypes. In order to define tolerance mechanisms and test the above hypotheses in humans, they have developed a novel system to track self-reactive B cells. This system utilizes a specific antibody variable region gene segment, VH4.34, as a surrogate marker of autoreactivity. Preliminary results support the hypothesis that B lymphocyte tolerance is defective in SLE and is in particular perturbed during the naive to memory B cell transition. Exactly where and how tolerance is abrogated during this transition remains to be defined. The present proposal will focus on further refining the distribution of autoreactive B cells into distinct subsets by flow cytometry and immunohistochemistry along the naive to memory transition in peripheral blood and lymphoid tissue from lupus patients in order to determine specifically where tolerance is abrogated. Aim 2 will ask what the mechanism of tolerance escape is by characterizing defects in signaling and function in lupus naive B cells compared to normal controls and correlating this with loss of tolerance. Signal transduction will be measured by calcium fluxes, tyrosine phosphorylation, and recruitment of downstream signaling pathways. Functional analysis will include determination of proliferative responses and expression of activation markers. Aim 3 will define what happens to the above abnormalities after treatment with a specific immunotherapy that targets B cells in a systematic fashion. By enumerating B cell subsets and characterizing defects in signal transduction and function in lupus B cells before and after B cell depletion, they will further elucidate the critical role of B cells in the immunopathogenesis of SLE. Given the long-term goal of understanding the basic mechanisms underlying the loss of immune tolerance, as an academic rheumatologist, this project and career award combined with the interactive research environment and the mentoring available at the University of Rochester is intended to foster the candidate's development as a basic science immunologist and independent investigator.